**3. Results**

*3.1. Comparison of Bacterial Composition in Unwashed Biopsies and Washed Biopsies*

3.1.1. A Decrease in Cultured Bacteria was Observed for the Washed Biopsies

The overall number of cultured non-*Helicobacter* species decreased in the washed biopsies compared to the unwashed biopsies, suggesting that many bacteria do not adhere to the tissue. A total of 27 biopsy pairs showed reduced or no growth in the washed biopsy compared to the growth observed in the unwashed biopsy (Table 2). Only 5 biopsy pairs showed unchanged growth, while 3 showed increased growth.


**Table 2.** Change in growth of bacterial species cultured from the biopsy pairs.

The total number of colonies of cultured *Streptococcus* spp. decreased, but its relative abundance was higher in the washed biopsies compared to the unwashed biopsies (Figure 2, Table 3). The cultured bacteria in the biopsies were dominated by *Streptococcus* spp., followed by *Rothia* spp. and *Actinomyces* spp. (Table 3).

1XPEHURIFRORQLHVFXOWXUHGIURP

**Figure 2.** The number of bacterial species isolated from culture of gastric cancer and dyspepsia patient biopsies. "Others" include *Bacillus* spp., *Corynebacterium* spp., *Enterobacter* spp., *Enterococcus* spp., *Haemophilus* spp, *Micrococcus* spp., *Neisseria* spp., and *Stenotrophomonas* spp.


**Table 3.** Distribution of total cultured bacterial groups.

### 3.1.2. Microbiome Analysis

Bacterial DNA was detected in all biopsies, even in those were no growth was observed. The ten most prevalent groups in the microbiome analysis were *Helicobacter* spp., *Streptococcus* spp., *Prevotella* spp., *Escherichia* spp., *Veillonella* spp., *Fusobacterium* spp., *Haemophilus* spp., *Rothia* spp., *Neisseria* spp., and *Alloprevotella* spp. (Figure 3). The average relative abundance of *H. pylori* increased in some of the washed biopsies compared to the unwashed, but this was not always observed in the individual samples (Table 4). The increase in *H. pylori* was therefore not significant. The "other bacteria" belong to over 100 di fferent bacterial groups, of which some species were only present in few biopsies. The bacterial groups that were found in several of the biopsies are among the genera: *Abiotrophia, Aggregatibacter, Atopobium, Campylobacter, Capnocytophaga, Catonella, Corynebaccterium, Dialister, Eubacterium, Filifacter, Flavobacterium, Gemella, Granulicatella, Lachnoanaerobaculum, Lactobacillus, Leptotrichia, Megasphaera, Oribacterium, Parvimonas, Peptostreptococcus, Porphyromonas, Propionibacterium, Selenomonas, Solobacterium, Staphylococcus, Stenotrophomonas, Stomatobaculum* and *Treponema* (Table 5). The relative abundance of other non-*H. pylori* bacteria was similar between unwashed and washed samples, and none of the listed groups showed a significant change in relative abundance in either dyspepsia patients (Figure 3a) or gastric cancer patients (Figure 3b). No significant di fferences were observed in the bacterial distribution between the patient groups. Comparison of the bacterial diversity within the samples showed no significant di fferences between the unwashed and washed biopsies (Figure 3c), and similar bacterial species were clustered in both groups (Figure 3d).


**Table 4.** Mean and standard error of the 10 most common bacterial groups as percentage of total bacterial reads in the microbiome analysis.

**Table 5.** Mean and standard error of the most common bacterial groups belonging to "other bacteria" as percentage of total bacterial reads in the microbiome analysis.

